Project Summary/Abstract Brain edema and associated intracranial hypertension are major and potentially lethal complications of acute liver failure (ALF) that have limited therapeutic options It is generally believed that ammonia is largely responsible for the edema in ALF, and that the edema is principally cytotoxic, i.e., due to astrocyte swelling. In addition to ammonia, emerging evidence also suggests other ALF-related toxins cytokines (CKs) and lipopolysaccharide (LPS), that are produced secondary to sepsis or liver necrosis, can generate an inflammatory response that may exacerbate the effect of ammonia on the astrocyte swelling and brain edema in ALF. Thus, ammonia and ALF-related toxins may directly impact astrocytes, or may influence other neural cells that indirectly contribute to astrocyte swelling. One likely candidate cell is the endothelial cell (EC), as this is the first cell to be affected by blood-borne ALF-related toxins (i.e., ammonia, LPS, CKs). Moreover, the close proximity of ECs to astrocytes places ECs in a strategic position whereby they can readily influence astrocytes under normal and pathological conditions. A major factor through which LPS and CKs influence ECs is the Toll-like receptor-4 (TLR4). However, the means by which ammonia impacts ECs is not known. Our overarching hypothesis is that (a) ammonia upregulates/activates TLR4 in ECs and such upregulation/ activation results in the generation of cell swelling mediators that contribute to the astrocyte swelling/brain edema in ALF, and (b) that LPS and CKs exacerbate the astrocyte swelling/brain edema in ALF through mechanisms similar to those of ammonia. In support of our hypothesis, we have generated preliminary data showing that the addition of conditioned media (CM) from ECs treated with ammonia results in astrocyte swelling; such swelling was markedly potentiated when CM from ECs treated with a combination of ALF- related toxins (ammonia, CKs, LPS) were added to astrocytes. Ammonia upregulated TLR4, whereas CM from ECs in which TLR4 was silenced and then added to astrocytes resulted a reduction in cell swelling, suggesting a critical role of endothelial TLR4 in the astrocyte swelling caused by CM from ECs. Likewise, transgenic mice (Tg) deficient in TLR4 were partially protected from the development of brain edema in ALF. Additionally, CM from ECs exposed to ammonia resulted in the upregulation of astrocytic Na+,K+, Cl- co-transporter (NKCC) and aquaporin-4 (AQP4), factors that represent penultimate events in the mechanism of astrocyte swelling/brain edema in ALF. The overall goal of this proposal is to investigate mechanisms by which ECs contribute to astrocyte swelling/brain edema in ALF and the critical role of TLR4 in that process. Aim #1 will examine the effect of ammonia in the upregulation/activation of TLR4; mechanisms by which ammonia upregulates TLR4, as well as potential additive/synergistic effects by other ALF-related toxins (LPS and CKs) on TLR4 upregulation. Aim #2 will examine the means by which TLR4 contributes to astrocyte swelling; the role of TLR4 in the activation of endothelial NF-6B; and the involvement of TLR4 in the formation and release of cell swelling mediators in ECs. Aim #3 will investigate mechanisms by which CM from ammonia-treated ECs cause astrocyte swelling by examining the effect of CM on activation of astrocytic NF-6B and regulation of NKCC and AQP4, the latter two representing penultimate factors in the mechanism of ammonia-induced astrocyte swelling. Aim #4 will establish which of the mechanisms identified in Specific Aims 1-3 that are involved in astrocyte swelling in culture, also contribute to the brain edema in vivo. Additionally, we will employ TLR4-KO mice to establish the role of endothelial TLR4 in the brain edema of ALF. We anticipate that a successful outcome of this proposal will not only advance our knowledge on the pathogenesis of the brain edema in ALF, but will also result in the identification of novel therapeutic targets aimed at alleviating this potentially lethal condition.